Correction: Do multiple experimenters improve the reproducibility of animal studies?

[This corrects the article DOI: 10.1371/journal.pbio.3001564.].

Do multiple experimenters improve the reproducibility of animal studies?

The credibility of scientific research has been seriously questioned by the widely claimed "reproducibility crisis". In light of this crisis, there is a growing awareness that the rigorous standardisation of experimental conditions may contribute to poor reproducibility of animal studies. Instead, systematic heterogenisation has been proposed as a tool to enhance reproducibility, but a real-life test across multiple independent laboratories is still pending. The aim of this study was therefore to test whether heterogenisation of experimental conditions by using multiple experimenters improves the reproducibility of research findings compared to standardised conditions with only one experimenter. To this end, we replicated the same animal experiment in 3 independent laboratories, each employing both a heterogenised and a standardised design. Whereas in the standardised design, all animals were tested by a single experimenter; in the heterogenised design, 3 different experimenters were involved in testing the animals. In contrast to our expectation, the inclusion of multiple experimenters in the heterogenised design did not improve the reproducibility of the results across the 3 laboratories. Interestingly, however, a variance component analysis indicated that the variation introduced by the different experimenters was not as high as the variation introduced by the laboratories, probably explaining why this heterogenisation strategy did not bring the anticipated success. Even more interestingly, for the majority of outcome measures, the remaining residual variation was identified as an important source of variance accounting for 41% (CI95 [34%, 49%]) to 72% (CI95 [58%, 88%]) of the observed total variance. Despite some uncertainty surrounding the estimated numbers, these findings argue for systematically including biological variation rather than eliminating it in animal studies and call for future research on effective improvement strategies.

Individualized social niches in animals: Theoretical clarifications and processes of niche change.

What are social niches, and how do they arise and change? Our first goal in the present article is to clarify the concept of an individualized social niche and to distinguish it from related concepts, such as a social environment and a social role. We argue that focal individuals are integral parts of individualized social niches and that social interactions with conspecifics are further core elements of social niches. Our second goal in the present article is to characterize three types of processes-social niche construction, conformance, and choice (social NC3 processes)-that explain how individualized social niches originate and change. Our approach brings together studies of behavior, ecology, and evolution and integrates social niches into the broader concept of an individualized ecological niche. We show how clarifying the concept of a social niche and recognizing the differences between the three social NC3 processes enhance and stimulate empirical research.

Social niche shapes social behavior and cortisol concentrations during adolescence in female guinea pigs.

Individualized social niches arise in social groups, resulting in divergent social behavior profiles among group members. During sensitive life phases, the individualized social niche can profoundly impact the development of social behavior and associated phenotypes such as hormone (e.g. cortisol) concentrations. Focusing on adolescence, we investigated the relationship between the individualized social niche, social behavior, and cortisol concentrations (baseline and responsiveness) in female guinea pigs. Females were pair-housed in early adolescence (initial social pair formation), and a social niche transition was induced after six weeks by replacing the partner with either a larger or smaller female. Regarding social behavior, dominance status was associated with aggression in both the initial social pairs and after the social niche transition, and the results suggest that aggression was rapidly and completely reshaped after the social niche transition. Meanwhile, submissive behavior was rapidly reshaped after the social niche transition, but this was incomplete. The dominance status attained in the initial social pair affected the extent of submissive behavior after the social niche transition, and this effect was still detected three weeks after the social niche transition. Regarding cortisol concentrations, higher baseline cortisol concentrations were measured in dominant females in the initial social pairs. After the social niche transition, cortisol responsiveness significantly increased for the females paired with a larger, older female relative to those paired with a smaller, younger female. These findings demonstrate that the social niche during adolescence plays a significant role in shaping behavior and hormone concentrations in females.

Conditional on the social environment? Roots of repeatability in hormone concentrations of male guinea pigs.

Individual differences in behavioral and physiological traits among members of the same species are increasingly being recognized as important in animal research. On the group level, shaping of behavioral and hormonal phenotypes by environmental factors has been reported in different taxa. The extent to which the environment impacts behavior and hormones on the individual level, however, is rather unexplored. Hormonal phenotypes of guinea pigs can be shaped by the social environment on the group level: pair-housed and colony-housed males differ systematically in average testosterone and stressor-induced cortisol levels (i.e. cortisol responsiveness). The aim of the present study was to evaluate whether repeatability and individual variance components (i.e. between- and within-individual variation) of hormonal phenotypes also differ in different social environments. To test this, we determined baseline testosterone, baseline cortisol, and cortisol responsiveness after challenge in same-aged pair-housed and colony-housed guinea pig males over a period of four months. We found comparable repeatability for baseline cortisol and cortisol responsiveness in males from both social conditions. In contrast, baseline testosterone was repeatable only in males from colonies. Interestingly, this result was brought about by significantly larger between-individual variation of testosterone, that was not explained by differences in dominance rank. Individualized social niches differentiated under complex colony, but not pair housing, could be an explanation for this finding.

Male social niche conformance? Effects of manipulated opportunity for extra-pair mating on behavior and hormones of male zebra finches.

Success in sperm competition is an important determinant of male fitness in mating systems with female multiple mating. Thus, sperm competition risk represents a key dimension of the male social environment to which individual males are expected to adaptively adjust their reproductive phenotype. Such adaptive phenotypic adjustment we here refer to as male social niche conformance. In this pre-registered study, we investigated how male zebra finches, Taeniopygia guttata, adjust their behavior to sperm competition risk. We experimentally manipulated the opportunity for extra-pair mating to create two levels of sperm competition risk: 1) Single-pair, no sperm competition risk; 2) Double-pair, sperm competition risk. We compared male courtship, mate guarding, copulation rates, and aggression between the treatment groups. To identify hormonal correlates of male behavioral adjustment, we measured plasma testosterone and corticosterone levels before and after the social treatment started. Contrary to our pre-registered predictions, males from the Double-pair treatment group decreased courtship rates compared to those from the Single-pair group, and Double-pair males responded less aggressively towards intruders than Single-pair males. Testosterone levels decreased over the breeding cycle, but social treatment had no effect on either testosterone or corticosterone levels. Our results indicate that male zebra finches do not intensify courtship or competitive reproductive behaviors, or upregulate key hormones when another breeding pair is present. Although we found no evidence for the predicted adaptive behavioral responses to sperm competition risk, we show that male zebra finches plastically adjust their behavior to their social environment.

Repeatability of endocrine traits and dominance rank in female guinea pigs.

BACKGROUND: Glucocorticoids (e.g. cortisol) are associated with variation in social behavior, and previous studies have linked baseline as well as challenge-induced glucocorticoid concentrations to dominance status. It is known that cortisol response to an acute challenge is repeatable and correlates to social behavior in males of many mammal species. However, it is unclear whether these patterns are also consistent for females. The aim of this study was to investigate whether baseline and response cortisol concentrations are repeatable in female guinea pigs (Cavia aperea f. porcellus) and whether dominance rank is stable and correlated to baseline cortisol concentration and/or cortisol responsiveness. RESULTS: Our results show that cortisol responsiveness (after 1 h: R = 0.635, 95% CI = 0.229, 0.927; after 2 h: R = 0.764, 95% CI = 0.433, 0.951) and dominance rank (R = 0.709, 95% CI = 0.316, 0.935) of females were significantly repeatable after six weeks but not correlated. Baseline cortisol was not repeatable (R = 0, 95% CI = 0, 0.690) and also did not correlate to dominance rank. Furthermore, the difference in repeatability estimates of baseline and response values was due to high within-individual variance of baseline cortisol concentration; the amount of between-individual variance was similar for baseline cortisol and the two measures of cortisol responsiveness. CONCLUSIONS: Females occupying different dominance ranks did not have long-term differences in cortisol concentrations, and cortisol responsiveness does not seem to be significantly involved in the maintenance of dominance rank. Overall, this study reveals the remarkable stability of cortisol responsiveness and dominance rank in a female rodent, and it remains an open question whether the magnitude of cortisol responsiveness is adaptive in social contexts for females.

Individuality meets plasticity: Endocrine phenotypes across male dominance rank acquisition in guinea pigs living in a complex social environment.

The time of dominance rank acquisition is a crucial phase in male life history that often affects reproductive success and hence fitness. Hormones such as testosterone and glucocorticoids can influence as well as be affected by this process. At the same time, hormone concentrations can show large individual variation. The extent to which such variation is repeatable, particularly in dynamic social settings, is a question of current interest. The aim of the present study was therefore to investigate how dominance rank and individual differences contribute to variance in hormone concentrations during male rank acquisition in a complex social environment. For this purpose, dominance rank as well as baseline testosterone, baseline cortisol, and cortisol responsiveness after exposure to a novel environment were determined in colony-housed guinea pig males from late adolescence through adulthood. Hormone-dominance relationships and repeatability of hormone measures beyond their relation to rank were assessed. There was a significant positive relationship between baseline testosterone and rank, but this link became weaker with increasing age. Baseline cortisol or cortisol responsiveness, in contrast, were not significantly related to dominance. Notably, all three endocrine parameters were significantly repeatable independent of dominance rank from late adolescence through adulthood. Baseline testosterone and cortisol responsiveness showed a significantly higher repeatability than baseline cortisol. This suggests that testosterone titres and cortisol responsiveness represent stable individual attributes even under complex social conditions.

Adaptive reshaping of the hormonal phenotype after social niche transition in adulthood.

Phenotypic plasticity allows individuals to adjust traits to the environment. Whether long-term adjustments of the phenotype occur during later life stages is largely unknown. To address this question, we examined whether hormonal phenotypes that are shaped by the environment during adolescence can still be reshaped in full adulthood. For this, guinea pig males were either housed in mixed-sex colonies or in heterosexual pairs. In adulthood, males were individually transferred to pair housing with a female. This way, a social niche transition was induced in colony-housed males, but not in pair-housed males. Before transfer, corresponding to findings in adolescence, adult colony-housed males showed significantly higher baseline testosterone levels and lower cortisol responsiveness than pair-housed males. One month after transfer, the hormonal phenotype of colony-housed males was changed towards that of pair-housed males: animals showed comparable baseline testosterone levels and cortisol responsiveness was significantly increased in colony-housed males. This endocrine readjustment builds the basis for an adaptive behavioural tactic in the new social situation. Thus, an adaptive change of the behavioural phenotype may still occur in adulthood via modification of underlying mechanisms. This suggests a greater role for developmental plasticity in later life stages than is commonly presumed.

Does the early social environment prepare individuals for the future? A match-mismatch experiment in female wild cavies.

BACKGROUND: The social environment that mothers experience during pregnancy and lactation has a strong effect on the developing offspring. Whether offspring can be adaptively shaped to match an environment that is similar to the maternal one is still a major question in research. Our previous work in wild cavies showed that females whose mothers lived in a stable social environment with few social challenges during pregnancy and lactation (SE-daughters) developed different behavioral phenotypes than females whose mothers lived in an unstable social environment with frequent social challenges during pregnancy and lactation (UE-daughters). In the present study we investigated whether SE-daughters are better adapted to a stable social environment, similar to their maternal one, than are UE-daughters, for which the stable social environment represents a mismatch with their maternal one. For this purpose, we established pairs of one UE- and one SE-daughter and housed them together under stable social conditions for one week. Dominance ranks, behavioral profiles, glucocorticoid levels, cortisol responsiveness and body weight changes were compared between the groups. We hypothesized that SE-daughters fare better in a stable social setting compared to UE-daughters. RESULTS: After one week of cohabitation in the stable social condition, UE-daughters had higher glucocorticoid levels, tended to gain less body weight within the first three days and displayed higher frequencies of energy-demanding behaviors such as rearing and digging than SE-daughters. However, there was no difference in cortisol responsiveness as well as in dominance ranks between UE- and SE-daughters. CONCLUSION: Higher glucocorticoid levels and less body weight gain imply that UE-daughters had higher energy demands than SE-daughters. This high energy demand of UE-daughters is further indicated by the increased display of rearing and digging behavior. Rearing implies increased vigilance, which is far too energy demanding in a stable social condition but may confer an advantage in an unstable social environment. Hence, SE-daughters seem to better match a stable social environment, similar to their maternal one, than do UE-daughters, who encountered a mismatch to their maternal environment. This data supports the environmental matching hypothesis, stating that individuals manage the best in environments that correspond to their maternal ones.

The adaptive shaping of social behavioural phenotypes during adolescence.

Developmental behavioural plasticity is a process by which organisms can alter development of their behavioural phenotype to be better adapted to the environment encountered later in life. This 'shaping' process depends on the presence of reliable cues by which predictions can be made. It is now established that cues detected by the mother can be used (primarily via hormones prenatally and maternal behaviour in the early postnatal stage) to shape the behavioural phenotype of her offspring. However, it is becoming increasingly clear that adolescence is another period in which conditions are well-suited for such shaping to occur. We review here how mammalian social behaviour may be shaped in adolescence. We identify limited extant examples, briefly discuss underlying mechanisms, and provide evidence that observed changes are indeed adaptive. We contend that while plasticity diminishes with age, the shaping of the behavioural phenotype in adolescence offers several advantages, including that adolescence is closer to the onset of mating than are earlier phases of life; that unlike earlier phases, information is obtained directly from the environment rather than mediated by the mother; and unlike later in adulthood, there is substantial underlying neural plasticity associated with development to support behavioural change. We also consider conditions that favour the occurrence of social behaviour plasticity during adolescence, including a high degree of sociality and a prolonged developmental period and the implication of these conditions for the occurrence of sex differences in the shaping process.

Adaptive shaping of the behavioural and neuroendocrine phenotype during adolescence.

Environmental conditions during early life can adaptively shape the phenotype for the prevailing environment. Recently, it has been suggested that adolescence represents an additional temporal window for adaptive developmental plasticity, though supporting evidence is scarce. Previous work has shown that male guinea pigs living in large mixed-sex colonies develop a low-aggressive phenotype as part of a queuing strategy that is adaptive for integrating into large unfamiliar colonies. By contrast, males living in pairs during adolescence become highly aggressive towards strangers. Here, we tested whether the high-aggressive phenotype is adaptive under conditions of low population density, namely when directly competing with a single opponent for access to females. For that purpose, we established groups of one pair-housed male (PM), one colony-housed male (CM) and two females. PMs directed more aggression towards the male competitor and more courtship and mating towards females than did CMs. In consequence, PMs attained the dominant position in most cases and sired significantly more offspring. Moreover, they showed distinctly higher testosterone concentrations and elevated cortisol levels, which probably promoted enhanced aggressiveness while mobilizing necessary energy. Taken together, our results provide the clearest evidence to date for adaptive shaping of the phenotype by environmental influences during adolescence.

Stress responsiveness and anxiety-like behavior: The early social environment differentially shapes stability over time in a small rodent.

The early social environment can profoundly affect behavioral and physiological phenotypes. We investigated how male wild cavy offspring, whose mothers had either lived in a stable (SE) or an unstable social environment (UE) during pregnancy and lactation, differed in their anxiety-like behavior and stress responsiveness. At two different time points in life, we tested the offspring's anxiety-like behavior in a dark-light test and their endocrine reaction to challenge in a cortisol reactivity test. Furthermore, we analyzed whether individual traits remained stable over time. There was no effect of the early social environment on anxiety-like behavior and stress responsiveness. However, at an individual level, anxiety-like behavior was stable over time in UE- but not in SE-sons. Stress responsiveness, in turn, was rather inconsistent in UE-sons and temporally stable in SE-sons. Conclusively, we showed for the first time that the early social environment differentially shapes the stability of behavioral and endocrine traits. At first glance, these results may be surprising, but they can be explained by the different functions anxiety-like behavior and stress responsiveness have.

Start early! Does social instability during the pre- and early postnatal development prepare male wild cavies for social challenge later in life?

BACKGROUND: The social environment the mother experiences during pregnancy and lactation can powerfully influence the offspring's behavioural profile. Our previous studies in wild cavies show that two different social environments during pregnancy and lactation bring about different behavioural strategies of male offspring later in life: An unstable social environment leads to a behavioural camouflage strategy, hypothesised to be beneficial at times of socially challenging situations. A stable social environment during early phases of life, however, leads to an early reproduction strategy, expected to be more successful at times of social stability. In the present study, we observed the behavioural strategies of the two types of males in direct comparison in a socially challenging situation: Two adolescent males were placed simultaneously in an unknown social group consisting of one adult male and two females in a semi-naturalistic environment. Cortisol as well as testosterone concentrations and activity levels were compared. Furthermore, paternities were analysed after the males reached sexual maturity. We hypothesised that sons showing a behavioural camouflage strategy are better adapted to cope with this socially challenging situation compared to those displaying an early reproduction strategy. RESULTS: At the beginning of the experiment, no differences in plasma cortisol concentrations between the males were found, both showed a highly significant increase due to the challenging situation. From day 5 until the end of the experiment (duration = 40 days) sons showing an early reproduction strategy had significantly higher plasma cortisol concentrations compared with those showing a behavioural camouflage strategy. Plasma testosterone concentrations did not differ significantly. Activity levels decreased significantly over time independently of the male's behavioural strategy. Both types of males did not sire offspring during the observation period. CONCLUSION: Higher cortisol values from day 5 until the end of the experiment in sons showing an early reproduction strategy indicate higher levels of stress in these males compared to those displaying a camouflage strategy. We conclude that the modulation of the males behavioural strategy due to an unstable social environment during early development facilitates the endocrine adaptation to a comparable social situation later in life.

Stability and change: Stress responses and the shaping of behavioral phenotypes over the life span.

In mammals, maternal signals conveyed via influences on hypothalamic-pituitary-adrenal (HPA) activity may shape behavior of the young to be better adapted for prevailing environmental conditions. However, the mother's influence extends beyond classic stress response systems. In guinea pigs, several hours (h) of separation from the mother activates not only the HPA axis, but also the innate immune system, which effects immediate behavioral change, as well as modifies behavioral responsiveness in the future. Moreover, the presence of the mother potently suppresses the behavioral consequences of this innate immune activation. These findings raise the possibility that long-term adaptive behavioral change can be mediated by the mother's influence on immune-related activity of her pups. Furthermore, the impact of social partners on physiological stress responses and their behavioral outcomes are not limited to the infantile period. A particularly crucial period for social development in male guinea pigs is that surrounding the attainment of sexual maturation. At this time, social interactions with adults can dramatically affect circulating cortisol concentrations and social behavior in ways that appear to prepare the male to best cope in its likely future social environment. Despite such multiple social influences on the behavior of guinea pigs at different ages, inter-individual differences in the magnitude of the cortisol response remain surprisingly stable over most of the life span. Together, it appears that throughout the life span, physiological stress responses may be regulated by social stimuli. These influences are hypothesized to adjust behavior for predicted environmental conditions. In addition, stable individual differences might provide a means of facilitating adaptation to less predictable conditions.

Domestication affects the structure, development and stability of biobehavioural profiles.

Domestication is an evolutionary process during which the biobehavioural profile (comprising e.g. social and emotional behaviour, cognitive abilities, as well as hormonal stress responses) is substantially reshaped. Using a comparative approach, and focusing mainly on the domestic and wild guinea pig, an established model system for the study of domestication, we review (a) how wild and domestic animals of the same species differ in behaviour, emotion, cognition, and hormonal stress responses, (b) during which phases of life differences in biobehavioural profiles emerge and (c) whether or not animal personalities exist in both the wild and domestic form. Concerning (a), typical changes with domestication include increased courtship, sociopositive and maternal behaviours as well as decreased aggression and attentive behaviour. In addition, domestic animals display more anxiety-like and less risk-taking and exploratory behaviour than the wild form and they show distinctly lower endocrine stress responsiveness. There are no indications, however, that domestic animals have diminished cognitive abilities relative to the wild form. The different biobehavioural profiles of the wild and domestic animals can be regarded as adaptations to the different environmental conditions under which they live, i.e., the natural habitat and artificial man-made housing conditions, respectively. Concerning (b), the comparison of infantile, adolescent and adult wild and domestic guinea pigs shows that the typical biobehavioural profile of the domestic form is already present during early phases of life, that is, during early adolescence and weaning. Thus, differences between the domestic and the wild form can be attributed to genetic alterations resulting from artificial selection, and likely to environmental influences during the pre- and perinatal phase. Interestingly, the frequency of play behaviour does not differ between the domestic and wild form early in life, but is significantly higher in domesticated guinea pigs at later ages. Concerning (c), there is some evidence that personalities occur in both wild and domestic animals. However, there may be differences in which behavioural domains - social and sexual behaviour, emotionality, stress-responsiveness - are consistent over time. These differences are probably due to changing selection pressures during domestication.

A comparison of brain gene expression levels in domesticated and wild animals.

Domestication has led to similar changes in morphology and behavior in several animal species, raising the question whether similarities between different domestication events also exist at the molecular level. We used mRNA sequencing to analyze genome-wide gene expression patterns in brain frontal cortex in three pairs of domesticated and wild species (dogs and wolves, pigs and wild boars, and domesticated and wild rabbits). We compared the expression differences with those between domesticated guinea pigs and a distant wild relative (Cavia aperea) as well as between two lines of rats selected for tameness or aggression towards humans. There were few gene expression differences between domesticated and wild dogs, pigs, and rabbits (30-75 genes (less than 1%) of expressed genes were differentially expressed), while guinea pigs and C. aperea differed more strongly. Almost no overlap was found between the genes with differential expression in the different domestication events. In addition, joint analyses of all domesticated and wild samples provided only suggestive evidence for the existence of a small group of genes that changed their expression in a similar fashion in different domesticated species. The most extreme of these shared expression changes include up-regulation in domesticates of SOX6 and PROM1, two modulators of brain development. There was almost no overlap between gene expression in domesticated animals and the tame and aggressive rats. However, two of the genes with the strongest expression differences between the rats (DLL3 and DHDH) were located in a genomic region associated with tameness and aggression, suggesting a role in influencing tameness. In summary, the majority of brain gene expression changes in domesticated animals are specific to the given domestication event, suggesting that the causative variants of behavioral domestication traits may likewise be different.

Effects of domestication on biobehavioural profiles: a comparison of domestic guinea pigs and wild cavies from early to late adolescence.

INTRODUCTION: Domestication can lead to marked alterations in the biobehavioural profile of a species. Furthermore, during ontogeny, the individual phenotype of an animal can be shaped by the environment in important phases such as adolescence. We investigated differences in biobehavioural profiles between domestic guinea pigs and their ancestor, the wild cavy, over the course of adolescence. At this age, comparisons between the two groups have not been conducted yet. Male guinea pigs and cavies were subjected to a series of tests twice: during the early and late phase of adolescence. We analysed emotional and social behaviours as well as cortisol reactivity and testosterone levels. RESULTS: Concerning emotional behaviour, cavies were more explorative and showed more anxiety-like behaviour in the open field test and dark-light test. They also were more risk-taking when having to jump off an elevated platform. Regarding social behaviour, cavies showed less social activity towards unfamiliar females and infants. Furthermore, while guinea pigs and cavies did not differ in basal cortisol levels, cavies showed distinctly higher and prolonged cortisol responses when exposed to an unfamiliar environment. Cavies also had lower basal testosterone titres. No substantial changes in biobehavioural profiles were revealed over the course of adolescence in both groups. CONCLUSIONS: Domestication led to a substantial shift in the biobehavioural profile of the guinea pig regarding all investigated domains in early and late adolescence. Hence, the differentiation between guinea pigs and cavies emerges early in ontogeny, well before the attainment of sexual maturity. The young individuals already show adaptations that reflect the differences between the natural habitat of cavies and the man-made housing conditions guinea pigs are exposed to. Higher levels of exploration and risk-taking and lower levels of anxiety-like behaviour are necessary for cavies in order to cope with their challenging environment. Their high cortisol reactivity can be interpreted as an energy provisioning mechanism that is needed to meet these demands. By contrast, guinea pigs are adapted to a less challenging environment with much higher population densities. Hence, their biobehavioural profile is characterised by higher levels of social activity and lower levels of exploration, risk-taking, and cortisol reactivity.

Stress response of fire salamander larvae differs between habitat types.

The larvae of the European fire salamander (Salamandra salamandra) can inhabit two different habitats: streams and ponds. Streams are characterized by lower predation risks and higher food availability. Thus, ponds are considered a less suitable habitat. To investigate the differential impacts of these two habitats on larval physiology, we measured the stress response of larvae. After successfully validating the measure of water-borne corticosterone release rates in fire salamander larvae, we measured the baseline and stress-induced corticosterone of 64 larvae from ponds and streams in the field. We found that larvae in ponds have a higher baseline and stress-induced corticosterone levels. Additionally, we performed a reciprocal transplant experiment (RTE) and tested whether larvae can adapt their stress responses to changing habitats. After two weeks, we did not find an increase in corticosterone levels when comparing stress-induced corticosterone values with baseline corticosterone values in larvae transferred into ponds, irrespective of their habitat of origin. However, larvae transferred into streams still exhibited an increase in the stress-induced corticosterone response in comparison with the baseline values. These results show that non-invasive hormone measurements can provide information on the habitat quality and potential adaptation and thus emphasize the potential for its use in conservation efforts.

The gut microbiota-immune-brain axis in a wild vertebrate: dynamic interactions and health impacts.

The gut microbiota-immune-brain axis is a feedback network which influences diverse physiological processes and plays a pivotal role in overall health and wellbeing. Although research in humans and laboratory mice has shed light into the associations and mechanisms governing this communication network, evidence of such interactions in wild, especially in young animals, is lacking. We therefore investigated these interactions during early development in a population of common buzzards (Buteo buteo) and their effects on individual condition. In a longitudinal study, we used a multi-marker approach to establish potential links between the bacterial and eukaryotic gut microbiota, a panel of immune assays and feather corticosterone measurements as a proxy for long-term stress. Using Bayesian structural equation modeling, we found no support for feedback between gut microbial diversity and immune or stress parameters. However, we did find strong relationships in the feedback network. Immunity was negatively correlated with corticosterone levels, and microbial diversity was positively associated with nestling body condition. Furthermore, corticosterone levels and eukaryotic microbiota diversity decreased with age while immune activity increased. The absence of conclusive support for the microbiota-immune-brain axis in common buzzard nestlings, coupled with the evidence for stress mediated immunosuppression, suggests a dominating role of stress-dominated maturation of the immune system during early development. Confounding factors inherent to wild systems and developing animals might override associations known from adult laboratory model subjects. The positive association between microbial diversity and body condition indicates the potential health benefits of possessing a diverse and stable microbiota.